Monoclonal antibody therapy for pancreas cancer

ABSTRACT

The present invention relates to the use of binding equivalents of monoclonal antibody 31.1, including chimerized and/or humanized versions thereof, antibody fragments as well as competitively binding and co-specific antibodies and antibody fragments, in the treatment of pancreatic cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/417,326, filed Jan. 27, 2017, which is a divisional of U.S. patentapplication Ser. No. 14/509,428, filed Oct. 8, 2014, now U.S. Pat. No.9,592,290, which is a divisional of U.S. patent application Ser. No.13/011,631, filed Jan. 21, 2011, now U.S. Pat. No. 9,034,588, which is adivisional of U.S. patent application Ser. No. 11/234,645, filed Sep.22, 2005, now abandoned, which is a continuation of U.S. patentapplication Ser. No. 10/472,008, filed Sep. 15, 2003, now abandoned,which is a national stage entry of International Pat. Appl. No.PCT/US2002.1009193, filed Mar. 15, 2002, which claims the benefit ofU.S. Provisional Appl. No. 60/276,284, filed Mar. 15, 2001, thedisclosures of each of which are hereby incorporated by reference intheir entireties.

SEQUENCE DISCLOSURE

This application includes as part of its disclosure a biologicalsequence listing which is being concurrently submitted through EFS-Web.Said biological sequence listing is contained in a file named“43282o2408.txt” which was created Oct. 17, 2018, and has a size of6,316 bytes, and is hereby incorporated by reference in its entirety.

1. INTRODUCTION

The present invention relates to the use of monoclonal antibody 31.1 andits equivalents and co-specific antibodies in the treatment of pancreascancer. It is based, at least in part, on the discovery that monoclonalantibody 31.1 is reactive with malignant, but not non-malignant,pancreatic cells. The present invention further provides polynucleotideand amino acid sequences comprising the light chain variable region andheavy chain variable region of Mu-31.1 as set forth in FIG. 2 and FIG.4, respectively. Such polynucleotide sequences may be used torecombinantly express 31.1 equivalent antibodies for use in the methodsof the invention.

2. BACKGROUND OF THE INVENTION 2.1. Pancreas Cancer

Pancreas cancer is the fifth leading cause of cancer death in the UnitedStates, with approximately 28,000 Americans expected to die from thedisease this year (Pancreas Cancer Web, The Johns Hopkins MedicalInstitutions, http://162.129.103.69: 80/PANCREAS-INTRO). At present, theonly potentially curative treatment is surgical removal of the cancer,in the context of an extensive and complex procedure which removes thehead, neck and uncinate process of the pancreas as well as the majorityof the duodenum (the“Whipple operation”). Without treatment, the overall5 year survival rate is only 3 percent (Id.).

Chemotherapy (often using gemcitabine (Gemzar®)) and radiation therapyare the main treatments offered to patients with unresectable tumors(Id.). An experimental immunotherapy is currently being studied in whicha patient's own cells are genetically modified to express the immunestimulatory protein, granulocyte macrophage colony stimulating factor,irradiated to prevent tumor growth, and then reintroduced into thepatient, where they will hopefully stimulate an immune response (1997,Cancer Res. 57: 1537-1546; Pancreas Cancer Web, The Johns HopkinsMedical Institutions, http://162.129.103.69:80/PANCREAS_MEDICAL_TX)).

2.2. Monoclonal Antibody 31.1

Antibody 31.1 represents a protein-directed monoclonal antibody derivedby immunizing BALB(c) mice with a preparation of membrane obtained frompooled (human) allogeneic colon carcinoma specimens. The cells used toprepare the antigen were fragmented using a nitrogen (Parr) bomb andthen subjected to ultracentrifugation. Membrane material was initiallytested by electron microscopy to guarantee consistency from batch tobatch, ruling out cytoplasmic and nuclear components. It was thensonicated and fractionated with sephadex G200. Discontinuouspolyacrylamide gel electrophoresis was used for the initial partialpurification (approximately 80%) and 30 μgm tested for delayed cutaneoushypersensitivity (DHR), (3). BALB mice were immunized by intraperitonealinjection of 50 micrograms of colon carcinoma associated antigen. Asecond injection was given 10 days later and the mice then sacrificed toobtain spleen cells for fusion. Fusion was performed by incubating 5×10⁷mouse spleen cells with 10⁷ sp2/0-AG 14 myeloma cells in 40% PEG. Theantigen defined by the monoclonal antibody 31.1 has been shown to haveM.W. of 72,000. Studies using immunoperoxidase have suggested that theantigen recognized by 31.1 is seen with greater frequency in the highergrade colon tumors. Specificity for the antibody is high, so that in astudy of shed colonocytes at the Mayo Clinic, sensitivity andspecificity were superior when compared with anti-CEA, anti-MUC1 andB72.3.

Several candidate antibodies were isolated and tested from the 1stgeneration TAA. All proved to be protein derived and relatively specificfor colon carcinoma. Antibody 31.1 corresponded to one of the twoantigens that have been shown to migrate closely on gel-electrophoresisand related to the immunogenic glycoprotein inducing the DHR. The murineversion is of IgG2a isotype which converts to an IgG1 isotype onchimerization. 31.1 was found to have strong localization indices. Assuch, this antibody was the first to be chimerized.

For chimerization of monoclonal antibody 31.1, the protein coding exonof 31.1 heavy chain variable region gene was spliced to the proteincoding exons of human gamma 1 chain constant region. PCR was employed.The 31.1 VH cDNA was amplified by the PCR using the degenerate backwardprimers synthesized based on the consensus first framework (FR1) regionDNA sequences and a forward primer synthesized according to theconsensus J-C junction region DNA sequences. The amplified 31.1 V_(H)DNA was cloned into the pBluescript vector and sequenced. Chimeric 31.1was produced by transfecting SP2/0 AG14 cells with the vector.

Monoclonal antibody 31.1 and a chimeric (humanized) version of thatantibody are described in U.S. Pat. No. 5,688,657 issued Nov. 18, 1997,now the subject of a reissue application, the contents of which arehereby incorporated by reference in its entirety herein. Furthermore,monoclonal antibody 31.1 has been deposited with the American TypeCulture Collection (“ATCC”), having an address at 10801 UniversityBlvd., Manassas, Va., 20110-2209 and assigned accession number ATCCPTA-2497 and the chimerized version has been deposited with the ATCC andassigned accession number 12316.

3. SUMMARY OF THE INVENTION

The present invention relates to the use of binding equivalents ofmonoclonal antibody 31.1, including chimerized and/or humanized versionsthereof, antibody fragments and competitively binding antibodies andantibody fragments, as well as co-specific antibodies, derivatives andfragments in the treatment of pancreas cancer.

It is based, at least in part, on in vitro studies using both murine andchimeric versions of 31.1 which compared the ADCC activites of the 31.1antibodies with D6-12 and 17.1a (Panorex). While the murine version of31.1 can induce a 35% ADCC response, the chimeric version has been shownto result in 80% of tumor cells being destroyed every three hours, usinga chromium release assay. This compares with a 30% rate of destructionassociated with D6-12 and a 15% rate for Panorex. Using xenograft modelswith human colon cancer cell lines LC-174T and Colo205, chimeric 31.1was found to cause regression of established tumor lines (well definedmolecules) after inoculating two million tumor cells into the hind legsof nude mice and administering intra-peritoneal antibody at 10 daysalong with human effector cells. At 30 days the volume of tumor in thetreated animals when compared to controls was reduced by more than 95%.Similar results may be expected when the antibody is directed towardpancreatic cancer cells, as the 31.1 antibody has been shown to bind toantigen present in pancreatic cancer cells, but not non-malignantpancreatic tissues.

The present invention provides polynucleotide and amino acid sequencescomprising the light chain variable region and heavy chain variableregion of Mu-31.1 which may be used to express chimerized 31.1antibodies. The nucleotide sequences of the invention include: (a) thenucleotide sequences shown in FIG. 2 (SEQ ID NOS:1 and 2) or FIG. 4 (SEQID NOS:4 and 5); (b) a nucleotide sequence (SEQ ID NOS:1 and 4) thatencodes the amino acid sequence shown in FIG. 2 (SEQ ID NO:3) or FIG. 4(SEQ ID NO:6); and (c) any nucleotide sequence that (i) binds to thenucleotide sequence of FIG. 2 or FIG. 4 under stringent hybridizationconditions, e. g., hybridization to filter bound DNA in 0.5 M NaHPO₄, 7%sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in0.1×SSC/0.1% SDS at 68° C. and (ii) encodes for a light and heavy chainvariable region capable of binding with the same immunospecificity asthe chimeric 31.1 monoclonal antibody.

The invention further provides for a new expression construct ofchimerized 31.1 antibody, termed pRc/CMV31.1 which has been depositedwith the ATCC and assigned accession no. ATCC [ ]. This plasmid carriesa dihydrofolate reductase (“dhfr”) expression unit driven by anenhancer-deficient SV40 early promoter that allows expression at greaterthan 200 mg/liter in dihydrofolate reductase deficient Chinese hamsterovary cells.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-F. Series of plasmids used to construct pRc/CMV 31.1 vector byinserting the Chi31.1-1 light chain and heavy chain genes into plasmidpDCM-dhfr (FIG. 1F).

FIG. 2. Nucleic acid sequence (double stranded, SEQ ID NOS:1 and 2) andpossible amino acid sequences (depending on reading frame, SEQ ID NO:3)of the 31.1 light chain variable region, showing restriction enzymecleavage sites.

FIG. 3. List of non-cutting enzymes of the light chain variable regionnucleic acid sequence shown in FIG. 2.

FIG. 4. Nucleic acid sequence (double stranded, SEQ ID NOS:4 and 5) andpossible amino acid sequences (depending on reading frame, SEQ ID NO:6)of the 31.1 heavy chain variable region, showing restriction enzymecleavage sites.

FIG. 5. List of non-cutting enzymes of the heavy chain variable regionnucleic acid sequence shown in FIG. 4.

FIG. 6A-B. An antibody-dependent cellular cytotoxicity (ADCC) assay wasconducted to test the effector function of the CHO Chi31.1 antibodyagainst target cells SW1643 and PANC-1. As cell lysis occurs in thepresence of 31.1 antibody (FIG. 6A) but not in the presence of controlantibody (FIG. 6B).

5. DETAILED DESCRIPTION OF THE INVENTION

For purposes of clarity of presentation and not by way of limitation,the detailed description is divided into the following two subsections:

i) monoclonal antibody 31.1 and its equivalents; and

ii) treatment protocol.

5.1. Monoclonal Antibody 31.1 and its Equivalents

Monoclonal antibody 31.1 is a murine monoclonal antibody (hereinafterreferred to as Mu-31.1), originally generated by immunization withpurified material from colon carcinoma cell membranes. Hybridoma cellssecreting this antibody have been deposited with the American TypeCulture Collection (“ATCC”) and assigned accession no. ATCC PTA 2497.

The present invention provides nucleic acid molecules and polypeptidescomprising the light chain variable region and-heavy chain variableregion of Mu-31.1. The nucleotide sequences of the invention include:(a) the DNA sequences shown in FIG. 2 or FIG. 4; (b) a nucleotidesequence that encodes the amino acid sequence shown in FIG. 2 or FIG. 4;(c) any nucleotide sequence that (i) hybridizes to the nucleotidesequence set forth in (a) or (b) under stringent conditions, e. g.,hybridization to filter-bound DNA in 0.5 M NaHP0₄, 7% sodium dodecylsulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.1×SSC/0. 1% SDS at68° C. (Ausubel F. M. et al., eds., 1989, Current Protocols in MolecularBiology, Vol. I, Green Publishing Associates, Inc., and John Wiley &sons, Inc., New York, at p. 2.10.3) and (ii) encodes a functionallyequivalent gene product. Functional equivalent gene products includethose polypeptides which compete with 31.1 for binding to its targetantigen. The invention also encompasses nucleotide sequences that encodepeptide fragments of the heavy and light chain variable regions, andfusion proteins thereof.

The nucleotides of the invention may be isolated using a variety ofdifferent methods known to those skilled in the art. For example, a cDNAlibrary constructed using RNA from cells or tissue known to express the31.1 monoclonal antibody or its equivalent, can be screened using alabeled nucleic acid probe derived from the sequences depicted in FIG. 2or FIG. 4. Further, nucleic acid sequences encoding the heavy and lightchain variable regions may be derived by performing PCR using twooligonucleotide primers designed on the basis of the nucleotidesequences disclosed herein. The template for the reaction may be cDNAobtained by reverse transcription of mRNA prepared from cell lines ortissue known to express the 31.1. monoclonal antibody.

The invention also encompasses (a) DNA vectors that contain any of theforegoing heavy and light chain variable region sequences and/or theircomplements (i. e., antisense); (b) DNA expression vectors that containany of the foregoing heavy and light chain variable region sequencesoperatively associated with a regulatory element that directs theexpression of the heavy and light chain variable region codingsequences; and (c) genetically engineered host cells that contain any ofthe foregoing heavy and light chain variable region sequencesoperatively associated with a regulatory element that directs theexpression of the coding sequences in the host cell. As used herein,regulatory elements include but are not limited to inducible andnon-inducible promoters, enhancers, operators and other elements knownto those skilled in the art that drive and regulate expression.

FIG. 2 shows the deduced amino acid sequence of the 31.1 light chainvariable region and FIG. 4 shows the deduced amino acid sequence of the31.1 heavy chain variable region. Thus, the amino acid sequences of theinvention include the amino acid sequence shown in FIG. 2 and FIG. 4.

The invention also encompasses proteins that are functionally equivalentto proteins encoded by the nucleotide sequences described above, asjudged by any of a number of criteria, including but not limited to theability to bind to the epitope recognized by the 31.1 monoclonalantibody.

Peptides corresponding to one or more domains of the heavy and lightchain variable regions, as well as fusion proteins in which the fulllength or a portion of the heavy and light chain variable region isfused to an unrelated protein are also within the scope of the inventionand can be designed on the basis of the nucleotide and amino acidsequences disclosed herein (see, FIG. 2 and FIG. 4).

While the heavy and light chain variable regions can be chemicallysynthesized (e. g., see Creighton, 1983, Proteins: Structures andMolecular Principles, W. H. Freeman & Co., N. Y.), the regions may beadvantageously produced by recombinant DNA technology using techniqueswell known in the art for expressing a nucleic acid containing heavy andlight chain variable region gene sequences and/or coding sequences. Suchmethods can be used to construct expression vectors containing thenucleotide sequences described above and appropriate transcriptional andtranslational control signals. These methods include, for example, invitro recombinant DNA techniques, synthetic techniques, and in vivogenetic recombination. (See, for example, the techniques described inSambrook et al., 1989, supra, and Ausubel et al., 1989, supra).

A variety of host-expression vector systems may be utilized to expressthe nucleotide sequences of the invention. Where the heavy and lightchain variable regions are expressed as a soluble derivative and are notsecreted, the peptide or polypeptide can be recovered from the hostcell. Alternatively, where the heavy and light chain variable regionsare secreted the peptide or polypeptides may be recovered from theculture media.

The expression systems that may be used for purposes of the inventioninclude but are not limited to microorganisms such as bacteriatransformed with recombinant bacteriophage, plasmid or cosmid DNAexpression vectors containing nucleotide sequences encoding the 31.1heavy and light chain variable regions; yeast transformed withrecombinant yeast expression vectors containing nucleotide sequencesencoding for the 31.1 heavy and light chain variable regions ormammalian cell systems harboring recombinant expression constructscontaining promoters derived from the genome of mammalian cells or frommammalian viruses.

Appropriate expression systems can be chosen to ensure that the correctmodification, processing and sub-cellular localization of the heavy andlight chain variable region protein occurs. To this end, host cellswhich possess the ability to properly modify and process antibodies forsecretion are preferred. For long-term, high yield production ofrecombinant proteins, such as that desired for development of cell linesfor production of chimeric antibodies, stable expression is preferred.Rather than using expression vectors which contain origins ofreplication, host cells can be transformed with DNA controlled byappropriate expression control elements and a selectable marker gene, i.e., tk, hgprt, dhfr, neo, and hygro gene, to name a few. Following theintroduction of the foreign DNA, engineered cells may be allowed to growfor 1-2 days in enriched media, and then switched to a selective media.

A chimeric version of murine 31.1, referred to hereinafter as Chi31.1-1,comprising variable region from Mu-31.1 together with human constantregion immunoglobulin sequences, is produced by hybridoma cellsdeposited with the ATCC and assigned accession no. ATCC CRL-12316.

In a specific embodiment of the invention, a second chimeric version ofmurine 31.1, hereinafter referred to as Chi31.1-2, having variableregions from Mu-31.1 and human constant immunoglobulin regions andderived from the Chi31.1-1 heavy and light chain genes, may be producedby expression of vector pRc/CMV31.1, described herein, and as shown inFIG. 1. This vector has the advantage of producing high yields ofchimeric antibody. A description of the preparation of this vector isprovided in the example section, below.

In specific non-limiting embodiments of the invention, further chimericversions may be produced comprising the variable regions of Mu-31.1. Forexample, the heavy chain variable region and light chain variable regionmay be generated using PCR primers designed based on the variable regionsequences set forth in FIG. 2 (light chain variable region) and FIG. 4(heavy chain variable region) or variants thereof to alter the terminito facilitate splicing in a vector of choice and using, as a source oftemplate DNA, DNA collected from a hybridoma that produces a 31.1-Abequivalent, such as one of the hybridomas set forth above which havebeen deposited with the ATCC. The variable region encoding sequences maythen be combined with human constant-region encoding sequences toproduce “humanized” antibody.

Alternatively, nucleic acid encoding Chi31.1-1 heavy and light chains(including human constant regions) may be inserted into variousexpression vectors to facilitate expression. Specific non-limitingexamples of such PCR primers are:

a) for insertion of Chi31.1-1 light chain encoding sequences at aBamHl/XbaI insertion site:

-   -   i) Chi31.1-LcBamHl (S):

5′-ATA GGA TCC ATG AAG TCA CAG ACC CAG GTC TTC G-3′ (SEQ ID NO:7)

-   -   ii) Chi31.1-LcXBaI (A):

5′-TTT CTA GAC TAA CAC TCT CCC CTG TTG AAG C-3′ (SEQ ID NO:8)

b) for insertion of Chi31.1-1 heavy chain encoding sequences at aEcoRI/NotI insertion site:

-   -   i) Chi31.1-HcEcoRI (S):

5′-ATA GAA TTC ATG GCT TGG GTG TGG ACC TTG CT-3′ (SEQ ID NO:9)

-   -   ii) Chi31.1-HcNotI (A): 5′-TTG CGG CCG CTC ATT TAC CCG GAG-3′        (SEQ ID NO:10). Such primers may be used in polymerase chain        reactions using, as template, DNA prepared from hybridoma cells        deposited with the ATCC and assigned accession no. ATCC        CRL-12316.

“Equivalents” of Mu-31.1 are defined herein as immunoglobulin moleculesor fragments or derivatives thereof which compete with Mu-31.1 forbinding to its target antigen, as evaluated using standard techniques.Such equivalents may include complete antibody molecules (i. e., havingtwo heavy chains and two light chains), single chain antibody molecules(see, for example, Lee et al., 1999, Molec. Immunol. 36: 61-71,incorporated by reference herein), fragments such as F (ab) and F (ab) 2fragments of Mu-31.1, Chi31.1-1, Chi31.1-2, or equivalent completeantibody molecules, and derivative molecules including, but not limitedto, one or more of the foregoing immunoglobulin molecules or fragmentsconjugated to a bioactive agent, or modified to more closely resemble ahuman immunoglobulin molecule (see, for example; Ryu et al., 1996, HumanAntibod. Hybridomas 7: 113122). Such equivalents, which include Mu-31.1,Chi31.1-1, Chi31.1-2, are collectively referred to as “31.1-Abequivalents”.

The use of co-specific antibodies and their equivalents (withequivalents having the same scope as that applied to the 31.1 antibody)is also envisioned according to the invention. A co-specific antibody toMu-31.1 (referred to as “31.1 co-specific antibodies”) may oralternatively may not compete with binding of Mu-31.1, but recognizes(i. e., binds to) the same target antigen, referred to herein as“31.1-Ag”). The co-specific antibodies to 31.1 and their equivalents arereferred to herein as “31.1 co-specific antibody equivalents”.

Any 31.1 antibody equivalent or 31.1 co-specific antibody equivalent tobe used in humans preferably has a structure which itself does notprovoke a deleterious immune reaction in humans. For example, said 31.1antibody equivalent or 31.1 co-specific antibody equivalent mayinherently lack such immunogenic structures or may be the product of a“humanization” process by standard techniques to minimize or eliminatestructures which would be recognized as non-self by a subject (e. g.chimerization and/or site by site engineering). 31.1-Ag appears to belocalized to the membrane of colon and pancreas cancers. Its presencehas not been detected on normal human tissue obtained fresh andimmediately frozen (TABLE A).

TABLE A Cross-reactivity to normal fresh frozen human tissues. Tissue(number) Staining paraffin Staining frozen samples Colon (3) Negative(3) Negative (2) Trace positive (1) Small bowel (3) Negative (3)Negative (3) Esophagus (3) Negative (3) Negative (3) Oral Mucosa (2)Negative (2) Negative (2) Jejunum (1) Negative (1) Negative (1) Stomach(1) Negative (1) Negative (1) Liver (3) Negative(3) Negative(3) Pancreas(3) Negative (3) Negative (3) Thymus (3) Negative (3) Negative(3) Heart(2) Negative (2) Negative(2) Prostate (2) Negative (2) Negative (2)Breast (3) Negative (3) Negative (3) Testis (1) Negative (1) Negative(1) Ovary (2) Negative (2) Negative (2) Salivary gland (3) Negative (3)Negative (3) Spleen (2) Negative (2) Negative (2) Brain (3) Negative (3)Negative (3) Lymph node (2) Negative (2) Negative (2) Adrenal (1)Negative (1) Negative (1) Vagina (1) Negative (1) Negative (1) WBC (1)Negative (1) Negative (1)

31.1-Ag is, however, found on the surface of colon and pancreas cancersobtained fresh at the moment of surgery and frozen (TABLE B).

TABLE B Localization of 31.1 antigen on colon and pancreas cancersStaining Cancer (number) Staining paraffin frozen samples Adenocarcinomaof colon Positive (3) Positive (3) Adenocarcinoma of pancreas (3)Positive (3) Positive (3)

It should be noted that this result differs from that presented in Table2 of U.S. Pat. No. 5,688,657 (at column 24, lines 1-26), which indicatesthat antibody Mu-31.1 did not bind to either of two pancreas tumorsamples tested. Table 1 of U.S. Pat. No. 5,688,657 (at column 23 lines1-38) shows that Mu-31.1 reacted with two out of three pancreaticcancer-derived cell lines. Based on the information contained in U.S.Pat. No. 5,688,657, one may have concluded that 31.1 Ag only appearedafter passage of the cells in culture, and was not present on freshpancreatic cancer tissue. It is therefore unexpected, based on thedisclosure of U.S. Pat. No. 5,688,657, that 31.1-Ag would be present on3/3 pancreatic tumor samples, as set forth in TABLE B herein.

Mu-31.1 is secreted from a hybridoma cell line developed by fusion withthe murine SP2 cell line cell-line. Mu-31.1, Chi31.1-1, and Chi31.1-2,31.1-Ab equivalents, and 31.1 co-specific antibodies may bemanufactured, for example and not by way of limitation, for clinical useby standard in vitro cell culture and downstream purification processes.For example, hybridoma cells may be grown in Geneticin (0.2 mg/ml) sincethe presence of the antibiotic has been observed to allow the hybridomacells to grow better.

Preferably, compositions comprising the forgoing 31.1-Ab equivalents and31.1 co-specific antibodies may be made without the addition of humanadditives. For example, the preparations may be filtered through abacterial Millipore 0.2 micron filter to eliminate contaminants andverified as sterile for bacteria and fungi by streaking blood agarplates and culture media with positive controls for 14 days. Thepreparation may be determined to be free of Mycoplasma by, for example,PCR Mycoplasma assays and by Mycoplasma Agar plates (Life Technology cat#18042-010) and Myco Test Kit (Life Technology Cat #15672-017) using 3T6control cells.

Media containing one or more of the foregoing 31.1-Ab equivalents or31.1 co-specific antibodies may be filtered through a Pall endotoxinfilter and the glassware heat sterilized to eliminate endotoxin.Desirably, but not by way of limitation, an appropriate endotoxin levelmay be 0.125 units/ml or less, as measured by the BioWhittaker Pyrogent03,250 test kit.

In preferred, non-limiting embodiments of the invention, one of theforegoing preparations may be treated so as to inactivate virus. Forexample, retrovirus may be inactivated by acetic acid treatment at pH 3for one hour during column chromatography and filtration through a PallUltipor Grade DV50 Virus Removal Filter of 10-40 nm.

In a specific, non-limiting embodiment of the invention, 50 mg ofCh31.1-1 is contained in a vial at a concentration of 2 mg/ml inphosphate buffered saline (“PBS”).

5.2. Treatment Protocols

The present invention provides for the use of 31.1-Ab equivalents and/or31.1 co-specific antibody equivalents, used singly or in combination, inthe treatment of pancreas cancer in a subject in need of such treatment.The method involves administering, to the subject, a therapeuticallyeffective dose of one or more 31.1-Ab equivalent and/or 31.1 co-specificantibody equivalent. A therapeutically effective dose is defined,herein, as a dose which achieves one or more of the following in thesubject: produces detectable pancreatic carcinoma cell lysis in thesubject; causes a decrease in the growth, or invasiveness, or size of apancreas tumor; causes an improvement in clinical symptoms; and/orcauses an increase in survival time. Preferably, but not by way oflimitation, a single dose of 31.1-Ab equivalent and/or 31.1 co-specificantibody equivalent may range from about 25 mg to about 1000 mg, andpreferably from about 100 mg to 250 mg. The magnitude of the dose may beadjusted on a patient-by-patient basis to avoid undesirable side effectsand/or toxicity. It is preferred that the 31.1-Ab equivalent and/or 31.1co-specific antibody equivalent is administered as a series (plurality)of single doses, administered at intervals of between about 1 and 4weeks, preferably every two weeks, until side effects rise to anundesirable level or disease progresses to an undesirable level. The31.1-Ab equivalent and/or 31.1 co-specific antibody equivalent may beadministered via any standard route; preferably, to test whether apatient tolerates the formulation (i. e., the patient does not manifestan undesirable allergic and/or other toxic reaction), it may first beadministered subcutaneously, and once adequate tolerance is shown, itmay be administered intravenously.

In one specific, non-limiting example, a protocol according to theinvention may be as follows.

Using aseptic procedures, a “humanized” 31.1-Ab equivalent and/or 31.1co-specific antibody equivalent, produced using standard biotechnologytechniques, may be filtered through a 0.22 micron low protein filterinto a glass infusion bottle or non-DEEP-containing infusion bagcontaining 0.9% sodium chloride to a final concentration of 0.4 mg/ml.The infusate may be mixed gently. If the infusion is observed to becloudy, it should not be administered.

To determine whether a patient tolerates treatment with the “humanized”31.1-Ab equivalent or 31.1 co-specific antibody equivalent, the patientmay be pre-medicated with diphenhydramine 25 mg i.v. and paracetamol 650mg p.o., and then 30 micrograms of 31.1-Ab equivalent or 31.1co-specific antibody equivalent may be injected subcutaneously. If noallergic toxicity or a grade 1 allergic toxicity occurs, intravenoustreatment will proceed. If a grade 1 allergic toxicity occurs,resolution of the toxicity will be necessary prior to proceeding withthe intravenous injection.

If the patient tolerates the subcutaneous test dose described in thepreceding paragraph, the patient may be treated with a first infusion of25 mg of the 31.1-Ab equivalent or 31.1 co-specific antibody equivalentover 2 hours. Premedication in the form of diphenhydramine 25 mg i.v.and paracetamol 650 mg p.o. may be given. The patient may then beobserved for any potential side effects for 6 hours after the injection.The patient may be monitored with vital signs prior to the injection,and every 15 minutes during the first hour of treatment, every 30minutes for two hours thereafter, and every hour thereafter until 6hours after completion of the infusion.

If the first infusion has been found to be tolerated, after 2 weeks, thepatient may then receive an infusion of 50 mg of the 31.1-Ab equivalentor 31.1 co-specific antibody equivalent, in a volume of 100 cc PBS orother suitable diluent, over 4 hours using the same clinical protocol asset forth in the preceding paragraph. If this second infusion has alsobeen found to be tolerated, the patient may then receive infusions of100 mg of the 31.1-Ab equivalent or 31.1 co-specific antibody equivalentin 100 cc diluent over 4 hours every two weeks, using theabove-described protocol. The patient may then continue such treatmentuntil intolerance develops or progression of disease occurs, andpreferably for a maximum of 4 months. If any grade 3 or higher toxicityoccurs due to the treatment, the patient may discontinue treatmentpermanently. If it is deemed that the toxicity is not treatment related,the patient may be able to resume treatment upon recovery of thetoxicity. If any grade 2 toxicity occurs during or after treatment, theinfusion may desirably be stopped. If recovery to grade 0 occurs, theinfusion may then be restarted. If recovery has not occurred by the timeof the next planned treatment, treatment may be delayed until recoveryto grade 0 has occurred. If recovery to grade 0 does not occurred within4 weeks, treatment may be discontinued permanently. If any allergicreaction of grade 2 or higher occurs, the treatment may be stopped andpreferably no further infusion may be given.

In specific non-limiting embodiments of the invention, the following mayserve as criteria for patients suitable for treatment:

a) the patient may suffer from a histologically confirmed recurrent ormetastatic adenocarcinoma of the pancreas, where the tumor reacts withthe 31.1-Ab equivalent or 31.1 co-specific antibody intended to be used;

b) treatment of the patient by a standard regimen for metastaticpancreas cancer may have failed;

c) disease in the patient may be measurable by one or more of thefollowing:

-   -   i) physical examination;    -   ii) computerized tomography or other radiological study;    -   iii) CEA levels; and/or    -   iv) Ca 19-9 levels;

d) the patient may be 18 years of age or older;

e) the patient may exhibit a WHO performance status of 0, 1, or 2;

f) the prognosis of the patient may indicate a life expectancy of atleast 12 weeks;

g) hematological testing of the patient may indicate the followingvalues:

-   -   i) WBC>3,000;    -   ii) HGB>10; and    -   iii) platelets>100,000;

h) clinical chemistry values may be as follows:

Creatinine, bilirubin, aspartate transaminase, alanine transaminase,alkaline phosphatase, and bilirubin are all less than or equal to 2times upper limit of normal; and/or

i) the patient has adequate peripheral venous access for repeated bloodsampling.

In specific non-limiting embodiments of the invention, the following mayserve as criteria for excluding patients who may be unsuitable fortreatment:

a) less than 4 weeks may have elapsed since prior chemotherapy (or 6weeks for nitrosoureas or mitomycin-C), since treatment with biologicalresponse modifiers or since radiation therapy;

b) the patient is currently receiving steroid therapy

c) the patient is pregnant (men and women on the study, if fertile, arecounseled to practice effective contraception);

d) the patient is a lactating female;

e) the patient suffers from a debilitating non-malignant co-morbidcondition, such as active infection or an acute intercurrentcomplication of malignancy;

f) there is central nervous system involvement;

g) the patient has previously received a bone marrow or other organtransplant;

h) the patient has a history of another malignancy, except foradequately treated non-melanoma cancer of the skin or in situ cancer ofthe cervix;

i) the patient has previously been exposed to murine monoclonal orpolyclonal antibodies; and/or

j) the patient is known to be HIV positive.

During the course of the study, non-limiting examples of adversereactions include shortness of breath, hypotension, cyanosis, rash,bronchospasm, chills, rigors, back pain, fever, cyanosis, nausea,vomiting, palpitations or any other adverse reaction.

In non-limiting embodiments of the invention, the following laboratorytests may desirably be performed to evaluate patients being treated bythe protocol. With regard to hematology tests, a complete blood count,differential, and platelet count may be obtained prior to each infusionand weekly during treatment until four weeks after the last injection.With regard to clinical chemistry tests, a complete chemistry panelmeasuring glucose, sodium, potassium, bicarbonate, chloride, blood ureanitrogen, creatinine, uric acid, calcium, inorganic phosphate, totalprotein, albumin, lactate dehydrogenase, aspartate transaminase, alaninetransaminase and alkaline phosphatase may be obtained weekly duringtreatment and until four weeks after the last injection. With regard tospecial laboratory tests, serum samples obtained from 10 cc of blood maybe collected before and within two minutes of each injection, at times15 min, 30 min, 60 min, 2, 4, 24 and 72 hours after completion of thefirst injection and every two weeks thereafter prior to each injectionand until four weeks after the last treatment and processed for thedetection of administered 31-1-Ab equivalent and/or 31.1 co-specificantibody equivalent. These serum samples may then be used to determineADCC, antibody concentration, and the presence of human antibodiesdirected toward the administered antibody equivalent. Urinalysis may beperformed at enrollment and before each of the injection as well as fourweeks after the last injection, with microscopic examination performedon any abnormal specimens.

In various embodiments of the invention, the following safetyassessments may desirably be made. For each of infusion, vital signsincluding the temperature, pulse and blood pressure of the patient maybe obtained prior to and after each infusion. The pulse and bloodpressure may be recorded every fifteen minutes during the first hour ofinfusion and then every half hour for two hours, followed by hourlyuntil 6 hours after the completion of the infusion. Patients may beobserved and vital signs monitored until six hours after the completionof the infusion or until return to baseline of the vital signs.

An initial evaluation and subsequent evaluations of the patient'sresponse to treatment may be performed as follows. Tumor measurement maybe performed by physical examination and or standard or specialradiological studies such as chest X-ray, computerized tomography,magnetic resonance imaging, or ultrasound. If more than one measurablelesion exists, representative lesions should be measured. The longestperpendicular measurements of the representative lesions may be recordedprior to treatment and every eight weeks. Levels of Ca 19.9 may bemonitored regularly, for example monthly.

Preferably written informed consent is obtained for each patient to betreated. Each patient should be given a verbal description of thetreatment, its potential risks and benefits as well as alternativetreatments available, prior to signing the written consent.

During the course of treatment, blood products, antibiotics, antiemetic,analgesics or other medications for stable coexisting medical conditionsmay be administered as appropriate.

The treatment may be discontinued in a patient if there is evidence ofprogressive disease, if a serious or unexpected adverse reaction occurs,or for other medically appropriate reasons.

In addition to the therapeutic uses described herein, the 31.1antibodies and functional equivalents thereof may be used to diagnosepancreatic carcinoma in a subject. The diagnostic methods of theinvention are based on the discovery that the 31.1 antibody selectivelybinds to an antigen expressed in pancreatic carcinoma cells but notnormal cells.

In accordance with the invention, measurement of levels of monoclonalantibody 31.1 reactivity in samples derived from a subject can be usedfor the diagnosis of diseases such as pancreatic carcinoma. Thedetection of monoclonal 31.1 antibody reactivity in a sample from asubject can be accomplished by any of a number of methods. Preferreddiagnostic methods can involve, for example, immunoassays wherein 31.1reactive antigen is detected by their interaction with an 31.1monoclonal antibody. Immunoassays useful in the practice of theinvention include but are not limited to assay systems using techniquessuch as Western blots, radioimmunoassays, ELISA (enzyme linkedimmunosorbent assay), “sandwich” immunoassays, immunoprecipitationassays, precipitin reactions, gel diffusion precipitin reactions,immunodiffusion assays, agglutination assays, complement fixationassays, immunoradiometric assays, fluorescent immunoassays, protein Aimmunoassays, to name but a few.

A biological sample, such as pancreatic tissue or other biologicaltissue, is obtained from a subject suspected of having a particularcancer or risk for cancer. Aliquots of whole tissues, or cells, aresolubilized using any one of a variety of solubilization cocktails knownto those skilled in the art. For example, tissue can be solubilized byaddition of lysis buffer comprising (per liter) 8 M urea, 20 ml ofNonidet P-40 surfactant, 20 ml of ampholytes (pH 3.5-10), 20 ml of 2mecaptoethanol, and 0.2 mM of phenylmethylsulfonyl fluoride (PMSF) indistilled deionized water.

Immunoassays for detecting expression of the 31.1 reactive antigentypically comprise contacting the biological sample, such as a tissuesample derived from a subject, with the 31.1 monoclonal antibody underconditions such that an immunospecific antigen-antibody binding reactioncan occur, and detecting or measuring the amount of any immunospecificbinding by the antibody. In a specific aspect, such binding of antibody,for example, can be used to detect the presence and increased productionof 31.1 reactive antigen wherein the detection of the antigen is anindication of a diseased condition.

6. EXAMPLE: PREPARATION OF PRC/CMV VECTOR

The pRc/CMV vector was prepared using a series of plasmids, as depictedin FIG. 1A-F. The heavy and light chains of Chi31.1-1 were cloned intothe pCR vector (FIG. 1A) by TOPO (Topoisomerase I) cloning. Sequencesused for inserting the light and heavy chain sequences into the pCRvector by PCR are as follows:

a) for insertion of the Chi31.1-1 light chainencoding region at a BamH1/XbaI insertion site: i) Chi31.1-LcBamH1 (S):(SEQ ID NO: 7) 5′-ATA GGA TCC ATG AAG TCA CAG ACC CAG GTC TTCG-3′ii) Chi31.1-LcXBaI (A): (SEQ ID NO: 8)5′-TTT CTA GAC TAA CAC TCT CCC CTG TTG AAG C-3′b) for insertion of the Chi31.1-1 heavy chainencoding region at a EcoRI/NotI insertion site: i) Chi31.1-HcEcoRI (S):(SEQ ID NO: 9) 5′-ATA GAA TTC ATG GCT TGG GTG TGG ACC TTG CT-3′ii) Chi31.1-HcNotI (A): (SEQ ID NO: 10)5′-TTG CGG CCG CTC ATT TAC CCG GAG-3′

These were then cloned from the pCR vector into the pDCM-dhfr vector,such that the light chain encoding region was inserted at the BamHl/Xbalsite (under the control of the cytomegalovirus (“CMV”) promoter, and theheavy chain encoding region was inserted into the EcoRI/Notl site, underthe control of a second CMV promoter element (FIG. 1F).

The pDCM-dhfr vector was prepared using the series of steps set forth inFIGS. 1B-E. A series of vector constructions using some relatedcomponents are described in Ryu et al., 1996, Hum. Antibod. Hybridomas7: 113-122 (based on the pRc/CMV vector (Invitrogen); see, for example,page 115 and FIG. 4 of Ryu et al.); Jin et al., 1995, Virus Res. 38:269; and Lee et al., 1999, Molec. Immunol. 36: 61-71 (see, for example,FIG. 2 of that publication).

Basically, the pcDNA3 vector (Invitrogen) (FIG. 1B) was used as thebasis for the pDCM vector (FIG. 1C), in that digestion with pairs ofrestriction enzymes followed by re-ligation was used, in parallelpreparations, to destroy certain cleavage sites and maintain others invector downstream of the CMV promoter sequences. Specifically, as shownin FIG. 1C, digestion of pcDNA3 with first HindIII and BamHI, followedby religation and then digestion with Hot and Apia, followed byreligation, resulted in the preservation of Bestir, EcoRI, EcoRV, BstXI,and NotI sites downstream of the promoter; subsequent cleavage with BsmIlinearized the molecule between the ampicillin and neomycin resistancegenes (component 1). In parallel, digestion of pcDNA3 with Bstxl andNotI, followed by removal of the small fragment and re-ligation, removedthe BstXI, EcoRI, EcoRV, BstXI, and NotI sites and left the HindIII,KpnI, BamHI, XhoI, Xbal and ApaI sites intact; cleavage with PvuII andNruI gave rise to a fragment containing the CMV promoter, the preservedsites, and BGHpA (component 2). Component 2 was inserted between theends of component 1, resulting in pDCM, having two different insertionsites for genes downstream of two respective CMV promoter elements. Asshown in FIG. 1E, a dihydrofolate reductase gene (“dhfr”) from KC-dhfrmay then be inserted into pDCM (see Lee et al., 1999, Molec. Immunol.36:61-71) to produce pDCM-dhfr. Alternatively, as shown in FIG. 1D, thedhfr gene from KC-dhfr may be incorporated into pcDNA3, to producepCdhfr, which may then be engineered by methods analogous to those shownin FIG. 1C to produce the two CMV promoter/insertion site cassette.

The Chi31.1-1 heavy and light chain encoding sequences were then clonedfrom the pCR vector into pDCM-dhfr, to form pRc/CMV, which may betransfected into CHO dhfr-cells, after which expressed chimericimmunoglobulin molecules may be collected according to standardtechniques.

7. EXAMPLE: HUMAN IMMUNE RESPONSE TO CHI31.1-1

To determine whether the 31.1 chimeric antibodies are capable ofinducing an immune response, plasma was collected from a human subjectwho had been administered Chi31.1-1 chimeric monoclonal antibody. Thepresence of an immune reaction in the patient toward the chimericantibody was tested using the following assay.

96 well microtiter plates were coated with Chi31.1-1 antibody, using asolution which was 10 micrograms per milliliter, with 100 microlitersper well. A preparation of Chi31.1-1 was biotinylated. Then, eithercontrol plasma or patient plasma (50 microliters) was introduced intowells, and 50 microliters of the biotinylated Chi31.1-1 was added. Theplates were then incubated for ninety minutes at 37 degrees centigradeand then the wells were washed and streptavidin-horseradish peroxidaseconjugate was added. The wells were then washed three times. Then TMBsubstrate (3,3′,5,5′ tetramethyl benzidine) was added, and the plateswere incubated for 20 minutes. Stop solution was added, and the amountof reacted substrate was determined.

The results are presented in TABLE C, and are expressed in nanograms ofChi31.1-1 bound per milliliter of plasma. Results greater than 2-foldabove the pre-treatment baseline are considered to be positive.Non-specific baseline binding values from 3 healthy normal samples werefound to be 4 plus or minus 2 nanograms per milliliter. The standard wasdetermined by using goat anti-human IgGI coated wells with variousconcentrations of biotinylated Chi31.1-1 monoclonal antibody.

TABLE C Human Immune Response to Chi31.1-1 Monoclonal Antibody (HAMA)Time ng/ml bound 0 hour (pretreatment) 2 1 hour 3 2 hours 2 3 hours 3 4hours 3 5 hours 2 6 hours 3 Next day 4 1 week 3 2 weeks 5

8. EXAMPLE: ADCC ACTIVITY OF CHO CHI31.1 ANTIBODY

The following section describes experiments demonstrating that the CHOChi31.1 monoclonal antibody has biological activity associated withdestruction of tumors. Specifically, the antibody was shown to haveantibody-dependent cellular cytotoxicity (ADCC).

A four hour ¹¹¹In release assay was used to measure ADCC activity.Target cells were the colon tumor cell line SW1643 and pancreatic cancercell line PANC-1. UPC-10 was used as a control antibody. Target cellswere labeled with 50 μCi of ¹¹¹In-oxyquinoline for 15 minutes at roomtemperature. Target cells (1×10⁴) in 50 μl were added to 96-well plate.Ratios of effector to target cells of 100:1, 50:1 and 25:1 were assayedin the presence of CHO31.1 (1 mg/well). The plates were incubated forfour hours at 37° C. in a humidified atmosphere containing 5% CO₂.Supernatant was harvested for gamma counting with the use of SkatronHarvester frames. Experiments were carried out in triplicate. Specificlysis was calculated with the use of the following formula:

${\%\mspace{14mu}{lysis}} = {100 \times \frac{{observed}\mspace{14mu}{{release}({cpm})}\text{-}{spontaneous}\mspace{14mu}{{release}({cpm})}}{{total}\mspace{14mu}{release}\mspace{14mu}({cpm})\text{-}{spontaneous}\mspace{14mu}{{{release}({cpm})}.}}}$

As presented in FIGS. 6A and 6B, the CHO 31.1 antibody, but not thecontrol UPC-10 antibody, was capable of mediating antibody-dependentcellular cytotoxicity against the target cells.

Various publications are cited herein, the contents of which are herebyincorporated by reference in their entireties herein.

What is claimed is:
 1. A method of killing cells that express apancreatic carcinoma associated antigen, comprising delivering to saidcells an effective amount of a 31.1 monoclonal antibody, wherein saidantibody is specific for pancreatic cancer cells.
 2. The method of claim1, wherein said cells are killed in a subject or are cultured cells. 3.The method of claim 1, wherein: (a) the light chain of said antibody isencoded by the nucleotide sequence SEQ ID NO: 1, SEQ ID NO: 3, or ahumanized variant of SEQ ID NO: 1 or SEQ ID NO: 3; and (b) the heavychain of said antibody is encoded by the nucleotide sequence of SEQ IDNO: 4, SEQ ID NO: 6, or a humanized variant of SEQ ID NO: 4 or SEQ IDNO: 6; or wherein said chimerized antibody is expressed by a cell linedeposited under ATCC Accession Number CRL-12316.
 4. The method of claim1, wherein said antibody is a recombinant, chimerized, co-specific, orhumanized antibody.
 5. The method of claim 1, wherein said antibody isselected from the group consisting of a single chain antibody, a F(ab)fragment, and a F(ab)₂ fragment.
 6. The method of claim 1, wherein thelight chain of said antibody is encoded by the nucleotide sequence ofSEQ ID NO: 1, SEQ ID NO: 3, or a humanized variant of SEQ ID NO: 1 orSEQ ID NO: 3, and/or wherein the heavy chain of said antibody is encodedby the nucleotide sequence of SEQ ID NO: 4, SEQ ID NO: 6, or a humanizedvariant of SEQ ID NO: 4 or SEQ ID NO:
 6. 7. The method of claim 1,wherein said antibody or fragment is conjugated to a bioactive agent. 8.The method of claim 1, wherein said method further comprises physicalexamination or a radiological study to detect the presence of pancreaticcancer in said subject.
 9. The method of claim 8, wherein saidradiological study comprises a chest X-ray, computerized tomography,magnetic resonance imaging, or ultrasound.
 10. The method of claim 8,wherein said subject has recurrent or metastatic adenocarcinoma of thepancreas.
 11. The method of claim 8, wherein said subject has cancer oris at risk for cancer.
 12. The method of claim 3, wherein said antibodyis a chimerized or humanized antibody.
 13. The method of claim 3,wherein said antibody is selected from the group consisting of a singlechain antibody, a F(ab) fragment, and a F(ab)₂ fragment.
 14. The methodof claim 3, wherein the light chain of said antibody is encoded by thenucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, or a humanizedvariant of SEQ ID NO: 1 or SEQ ID NO: 3, and/or wherein the heavy chainof said antibody is encoded by the nucleotide sequence of SEQ ID NO: 4,SEQ ID NO: 6, or a humanized variant of SEQ ID NO: 4 or SEQ ID NO: 6.15. The method of claim 3, wherein said antibody or fragment isconjugated to a bioactive agent.
 16. The method of claim 3, wherein saidmethod further comprises physical examination or a radiological study todetect the presence of pancreatic cancer in said subject.
 17. The methodof claim 16, wherein said radiological study comprises a chest X-ray,computerized tomography, magnetic resonance imaging, or ultrasound. 18.The method of claim 3, wherein said antibody is administered to asubject having recurrent or metastatic adenocarcinoma of the pancreas.19. The method of claim 3, wherein said antibody is administered to asubject having cancer or at risk for cancer.